The mesoscopic scale is between microscopic and macroscopic scales. In life sciences, mesoscopic imaging allows scientists to record, track and study details of biological systems in the context of an organ, body part, or organism. Mesoscopic imaging techniques have been developed for medical and clinical research, such as drug delivery, cancer diagnosis, etc. Especially when combined with novel nanoparticles and organic dyes in the near-infrared spectral regime, the mesoscopic imaging can probe deeper parts of the animal body. Here, we describe a timeresolved mesoscopic imaging approach, which can image deep inside of the whole mouse noninvasively. In addition, it uses the FastFLIM technique to measure the lifetime of the fluorescent probe. Since the lifetime carries information about the probe’s local microenvironment such as temperature, pH, ion concentration, etc., the lifetime imaging map obtained by the FastFLIM-mesoscope allows tracking quantitative dynamics of the probes in the whole animal body. The technique can also be used for quantitative intrinsic NADH metabolism mapping for real time monitoring of mitochondrial function. Here, we will show mesoscopic-scale NADH imaging in an oral cancer model.
Photon upconversion is a nonlinear process in which the sequential of absorption of two or more photons leads to the anti-stoke emission. Different than the conventional multiphoton excitation process, upconversion can be efficiently performed at low excitation densities. Recent developments in lanthanide-doped upconversion nanoparticles (UCNPs) have led to a diversity of applications, including detecting and sensing of biomolecules, imaging of live cells, tissues and animals, cancer diagnostic and therapy, etc. Measuring the upconversion lifetime provides a new dimension of its imaging and opens a new window for its applications. Due to the long metastable intermediate excited state, UCNP typically has a long excited state lifetime ranging from sub-microseconds to milliseconds. Here, we present a novel development using the FastFLIM technique to measure UCNP lifetime by laser scanning confocal microscopy. FastFLIM is capable of measuring lifetime from 100 ps to 100 ms and features the high data collection efficiency (up to 140-million counts per second). Other than the traditional nonlinear least-square fitting analysis, the raw data acquired by FastFLIM can be directly processed by the model-free phasor plots approach for instant and unbiased lifetime results, providing the ideal routine for the UCNP photoluminescence lifetime microscopy imaging.
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